Project Summary The global impact of rickettsial diseases is highlighted by historical records, reemergence of fatal arthropod-borne spotted and typhus fever rickettsioses, and emergence of new pathogens. Obligate intracellular Rickettsia species infect arthropods and vertebrates throughout their lifecycles. The intracellular lifestyle and reductive, AT-rich genomes of Rickettsia spp. pose immense challenges to research centered on characterizing mechanisms of pathogenicity. Nevertheless, a transdisciplinary approach towards studying rickettsiae can overcome the near intractability of these bacteria to conventional genetic methodologies. This application employs such an approach, focusing on the Rickettsiales vir homolog (rvh) type IV secretion system (T4SS). Virtually nothing is known about how the rvh machine assembles to translocate effectors into host cells. This is largely due to unprecedented rvh gene family expansion. rvh contains three duplicate families (rvhB4, rvhB8, rvhB9): RvhB4,8,9-I are conserved relative to equivalents in other T4SSs, while RvhB4,8,9-II have evolved atypical features. Furthermore, rvh contains proliferated VirB6-like genes (rvhB6a-e), which are tandemly arrayed and contain large N- and C-terminal extensions. Remarkably, the significance of gene family expansion in regards to rvh structure and function has not previously been explored. In this proposal we will test the hypothesis that all Rvh proteins underpinned by gene family expansion form a structurally unique T4SS that orchestrates the complex rickettsial lifecycle (transmission between arthropod vector and vertebrate host). To test our hypothesis, we will define the role of gene duplication in rvh function (AIM 1) and characterize the extracellular function of RvhB6a (AIM 2). Under AIM 1, we will determine how conserved (RvhB4,8,9-I) and atypical (RvhB4,8,9-II) duplicates contribute to rvh structure and function. This work will involve a blend of gene/protein expression analyses in various host cell backgrounds, protein-protein interactions via screens and targeted approaches, functional characterization of RvhB4 ATPases, and antisense RNA technology to knock-down of RvhB4, 8, 9-II expression. Under AIM 2, we will describe the significance of VirB6- like proliferation by characterizing the lone Rickettsia RvhB6 protein known to be extracellular (RvhB6a). This work will involve a blend of gene/protein expression analyses in various host cell backgrounds, subcellular localization assays for RvhB6a domains, rvh effector co-localization with RvhB6a, and identification of potential host targets of RvhB6a extracellular domains. Collectively, these studies will allow us to decipher the manner by which the odd rvh machine operates to selectively translocate effectors into host cells throughout specific timepoints of the rickettsial intracellular lifecycle. Importantly, our proposed research is novel to Rickettsiology and will provide a valuable addition to the knowledgebase of rickettsial pathogenesis, making our work highly innovative. Upon successful completion of our work, we will be able to move forward with mutagenesis studies to characterize rvh secretion, as well as understand what aspects of the bizarre rvh architecture are amenable to drug targeting. Thus our proposal, while highly exploratory, is in line with our ultimate goal of developing therapeutics to combat fatal rickettsioses.